The present invention relates to a method for introducing a substance into a cell, and in particular to an efficient transfection method involving a low incidence of cell-death. The invention also relates to kits for introducing a substance into a cell.
Throughout this text, the introduction of foreign substances, such as nucleic acid protein, peptides or other biological molecules into cells is termed transfection. Transfection, particularly of genetic material, has recently proved to be one of the most important techniques in molecular biology, particularly in relation to genetic engineering and protein engineering. The technique has allowed foreign DNA to be expressed in cells. This is of scientific interest in studying gene transcription and has a wide range of commercial applications involving expressing commercially useful gene products in convenient types of cell. More recently there has been interest in introducing both proteins and drugs into living cells without damaging the cells. A significant problem to be overcome when developing such techniques is the general imperviousness of the cell membrane. The cell membrane is normally impervious to even small molecules, unless they are very lipophilic. Even short-term damage to the cell membrane to render it more permeable tends to result in cell-death. This is a particular problem associated with electroporation, discussed below.
A number of methods have been devised for transfecting cells with foreign DNA or other substances. Early methods involved binding DNA to particles such as diethylaminoethyl (DEAE) cellulose or hydroxyapatite and adding pre-treated cells which are capable of taking up particles containing DNA. These early methods are very inefficient, the level of transfection achievable being very low.
More recently methods have been developed which make use of liposomes loaded with DNA that can be fused with cells. A further technique involves subjecting cells, typically plant cells, to an electric shock which causes the formation of holes in the cells. This method is termed electroporation.
In Biotechniques, Vol 17 No. 6 1994, page 118-1125, Clarke et al. disclose a method for introducing dyes, proteins and plasmid DNA into cells using an impact-mediated procedure. In this method, compressed gas is used to propel glass beads dispersed as a uniform aerosol onto adherent cells growing on a culture substratum. The impact of beads on the cells creates plasma membrane wounds. Molecules such as dyes, proteins and plasmid DNAs diffuse from the extracellular environment directly into the cytoplasmic compartment of the cell through the wounds.
In Nucleic Acids Research, Vol. 18, No. 21, 1990, p.6464, the effect of the osmolarity of the transfection medium is studied in relation to electroporation methods. It was reported that the optimum osmolarity of the transfection medium for transfection by electroporation is around 300 mOsm.
A significant problem associated with the above treatments is that they are very inefficient. In addition, a large proportion of the cells are killed by the above treatments. Moreover, the treatments are not selective. In fact, no methods are presently available for the selective transfection of cells. Furthermore, in the method of Clarke et al, only a limited number of cells can be transfected in a single treatment.
An object of the present invention is to overcome the above drawbacks and to provide an efficient method of transfection with good cell survival rates and the possibility of selective transfection of a sub-population of cells in a sample. Accordingly, the present invention provides a method for introducing a substance into a cell, which method comprises:
(a) contacting the cell with a recognition agent to bind the recognition agent to a recognition site on the surface of the cell; and
(b) separating the recognition agent from the cell thereby forming a hole in the surface of the cell.
The method of the invention is conveniently referred to herein, as xe2x80x9cimmunoporationxe2x80x9d, although not all methods of the invention actually involve any immunological components or interactions.
The cell can be contacted with the substance to be introduced simultaneously with the formation of the hole in step (b), or alternatively in a subsequent separate step. Introduction of the substance may thus occur as a result of contact of the substance with the cell which contains one or more holes which facilitate passage of the substance into the cell. This said hole allows passage into the cell membrane or more preferably the cytoplasm of the cell.
The hole formed is typically transient in that it only exists for a very short period before the cell membrane again forms a substantially continuous layer. The important characteristic of the hole is that it enables the substance which it is intended to introduce into the cell to pass from the outside to the inside of the cell.
The invention will now be described in further detail by way of example only, with reference to the accompanying drawings, in which:
FIG. 1 shows the effect of the osmolarity of the transfection medium on the transfection of HL60 cells;
FIG. 2 shows confocal images of (A) promyelocytic HL-60 cells transfected with TMR-dextran using anti-CD71-coated Dynafect beads, (B) promyelocytic HL-60 cells transfected with TMR-dextran using anti-CD11b-coated Dynafect beads (negative control), (C) 3 day DMSO differentiated HL-60 cells transfected with TMR-dextran using anti-CD71-coated Dynafect beads (negative control), (D) 3 day DMSO differentiated HL-60 cells transfected with TMR-dextran using anti-CD11b-coated Dynafect beads; and
FIG. 3 shows FACS analysis of the expression of GFP in: (A) promyelocytic HL-60 cells transfected with pEGFP-C1 using anti-CD71-coated Dynafect beads, (B) HL-60 cells transfected with pEGFP-C1 using anti-CD71-coated Dynafect beads 1 day after induction with DMSO, (C) HL-60 cells transfected with pEGFP-C1 using anti-CD71-coated Dynafect beads 3 days after induction with DMSO, (D) promyelocytic HL-60 cells transfected with pEGFP-C1 using anti-CD11b-coated Dynafect beads, (E) HL-60 cells transfected with pEGFP-C1 using anti-CD11b-coated Dynafect beads 1 day after induction with DMSO and (F) HL-60 cells transfected with pEGFP-C1 using anti-CD11b-coated Dynafect beads 3 days after induction with DMSO.
The step (b) of the present method is preferably carried out in a liquid medium. In the present context this is termed an immunoporation medium. To maximise the efficiency and rate of transfection, the liquid medium used to transfect the cells typically has an osmolarity of from 0.1-4.0 times the osmolarity of the cells. In the context of the present invention, the osmolarity of the cell means the normal osmolarity of the untreated cell. Preferably, the liquid medium has an osmolarity of from 30-1200 mOsm. When the cells to be transfected are adherent, the osmolarity of the liquid medium is preferably less than the osmolarity of the cells, more preferably from 30-150, e.g. 40-100 mOsm and most preferably from 40-50 mOsm. When the cells to be transfected are in the form of a suspension, the osmolarity of the liquid medium is preferably greater than the osmolarity of the cells, more preferably from 700-1100 mOsm, more preferably still from 800-1000 mOsm and most preferably from 950-1000 mOsm. Thus non-isotonic conditions, relative to the osmolarity of the cells, allows for most efficient immunoporation and the liquid medium will therefore preferably not be isotonic or approximately isotonic having regard to the osmolarity of the cells.
The osmolarity of a liquid medium is a measure of the concentration of ions in the medium. The ions present in the immunoporation medium are not limited to a particular type of ion, provided that they do not inhibit transfection and can be tolerated by the cells. An immunoporation medium having the desired osmolarity may be formulated using 10 times concentrated Earl""s balanced salt solution (EBSS) (Earl, W. R., 1934, Arch. Exp. Zell. Forsch., Vol. 16, p. 116) containing nutrient factors as a base and diluting as required.
In the present invention, any recognition agent can be used as long as it is capable of binding with a recognition site on the cell surface. A recognition agent and/or a recognition site may comprise one or more molecules which together comprise the site of affinity binding, e.g. the recognition site may be a receptor complex to which a ligand may be bound. In the context of the present invention, such pairs of recognition agents and recognition sites capable of binding to form complexes are termed xe2x80x9crecognition pairsxe2x80x9d. Recognition pairs may be any pair of substances which have an affinity for one another, that is any pair of substances which are affinity binding partners e.g. bind to one another selectively under physiological pH and ionic conditions. Preferably recognition pairs are of the ligand/receptor type, i.e. pairs of agents in which the ligand has a structure that is specific for a given receptor (e.g. an antibody/antigen-type interaction). However, the pairs may also have a more conventional chemical binding mechanism such as a covalent, ionic, hydrophobic or hydrogen bonding interaction. Thus cell adhesion molecules are also envisaged as recognition pairs.
When the recognition pair has a ligand/receptor-type interaction, the recognition site on the cell surface may be either the receptor, or the ligand and the recognition agent may thus be either the ligand or the receptor.
In a preferred embodiment of the present invention, the recognition agent is selected from an antibody, an antigen, a growth factor, a growth factor receptor, a sugar, a sugar receptor, a hormone, a hormone receptor, a cell adhesion molecule, an enzyme, an enzyme substrate, a co-enzyme, a protein, a synthetic ligand, a synthetic receptor, a phage, a phage receptor, and a molecule capable of binding to any of the above. More preferably, the recognition pair employed in the present invention is selected from the following complexes:
(i) an antibody/antigen complex,
(ii) a growth factor/growth factor receptor complex,
(iii) a sugar/sugar receptor complex,
(iv) a hormone/hormone receptor complex,
(v) a complex formed from cell adhesion molecules,
(vi) an enzyme/substrate complex,
(vii) an enzyme/co-enzyme complex,
(viii) a lectin/lectin receptor complex,
(ix) a protein/organic molecule complex,
(x) a synthetic ligand/synthetic receptor complex, and
(xi) a phage/phage receptor complex.
When the recognition pair is an antibody/antigen complex, there is no particular limitation on the type of antibody or antigen used. The antibody may be polyclonal or monoclonal, synthetic (such as chimeric antibodies) or single chain antibodies. Fragments of antibodies, such as Fv or Fab fragments may also be used. The antigens may include antigen fragments and haptens. Preferably the receptor is an HLA Class I and the ligand is an anti Class I antibody. Antibodies can be species specific, provided that the appropriate epitope and antibody-type are selected, such that the antibody recognises only one particular epitope.
A growth factor/receptor recognition pair preferred in the present invention is the EGF(epidermal growth factor)/EGF receptor complex. This growth factor receptor is expressed at high levels in certain tumour cells, such that using the growth factor leads to enhanced transfection of such tumour cells.
The mannose/mannose receptor complex is a preferred sugar/sugar receptor complex in the present invention. The advantage of mannose as a recognition agent is that the mannose receptor is present on the surface of macrophage cells. Mannose is thus useful in selectively transfecting macrophage cells in the presence of cells which do not have a mannose receptor on their surface.
An example of a preferred hormone/hormone receptor complex employed in the present invention is the insulin/insulin receptor complex. Insulin is useful as a recognition agent, since the insulin receptor is present on the surface of liver cells. Insulin is thus useful in selectively transfecting liver cells in the presence of cells which do not have an insulin receptor on their surface.
Oligosaccharides are preferred in the present invention as cell adhesion molecules. Specific oligosaccharide/selectin complexes have the advantage of being selective for specific tissues, depending on the oligosaccharide/selectin complex employed.
One enzyme/substrate recognition pair envisaged for use in the present invention is the 5xe2x80x2-AMP(adenylic acid, or analogue)/5xe2x80x2nucleotidase complex. This complex is generic for all cells and could be employed to transfect all cells in a sample, when selectivity is not important. A preferred enzyme/co-enzyme recognition pair is the NAD+ (nicotinamide adenine dinucleotide)/dehydrogenase complex, genetically engineered so that the dehydrogenase is on the cell surface.
An example of a protein/protein-binding molecule complex for use in the present invention is the avidin/biotin complex. However, any molecule which, like biotin, (in particular an organic molecule) is capable of binding to one of the ligands or receptors described above will be useful in the present invention. In addition to a protein/protein binding molecule, a peptide/(peptide or protein) binding molecule could be employed. The peptide may be, for example, a peptide from a peptide library.
Synthetic ligands and synthetic receptors can, in a preferred embodiment of the present invention, be genetically modified (GM) ligands and receptors. These types of recognition pair are particularly useful, since the GM ligand can be designed to be specific to a particular GM cell having a GM receptor on it surface.
The recognition pairs are not limited to the specific examples listed above, and in general may include any biological effector molecules, of which hormones and growth factors are merely examples. Since the method of the invention may be used to selectively transfect cells bearing a particular recognition site, it will be appreciated that preferred recognition sites are those which a re present on a sub-set of cells and which are an indication of a particular cell type or an indicator of adherent cells e.g. tumour cells or modified cells expressing e.g. a mutant surface protein. The recognition sites expressed on the surface of cells of a discrete lineage or on abherant or diseased cells are preferred.
Given that the methods of the invention involve forming holes in the cell membrane, changes in the behaviour of the membrane can affect the efficiency of transfection. The fluidity of membranes is very temperature dependent, and it has been found using the standard mixing conditions (see the Examples) that the level of transfection which could be achieved at 10xc2x0 C. was significantly less than that obtained at room temperature (22xc2x0 C.). At 30xc2x0 C., although good levels of transfection could be achieved, there was also a large increase in the non-specific binding of the transfection molecules to the cell surface. Thus, generally, preferred transfection temperatures will be in the region of 18 to 28xc2x0 C.
The present method has many advantages. For example, when using an antibody, the antibody can be made selective for antigens specific to certain types of cells. Accordingly, in a cell population containing a number of different cells, specific types of cells can be targeted for transfection in preference to other types of cells. Moreover, there is no significant decrease in the viability of the cells, even when large amounts of cell surface are removed. The holes formed in the cells are transient, remaining open for a sufficient time to allow the influx of macromolecules into the cell, but re-sealing before the viability of the cell is compromised. For instance, using the method of the present invention, cell-death is generally less than 25% and often less than 5%. On the other hand, if electroporation is used cell-death can sometimes be as high as 90%.
In the present method, the recognition agent is separated from the cell by introducing a force between the cell and the recognition agent. In a preferred embodiment, the force is created by perturbing a liquid medium containing the cell and the recognition agent. However, the liquid medium may alternatively be allowed to flow across the surface of the solid on which the cell is isolated. The force set up is such that access to the inside of the cell is created by ripping or tearing a transient hole in the cell surface.
The hole in the cell surface may be a rip or tear in the cell membrane and may also include a hole formed by entirely removing a portion of the cell membrane. The recognition agent alone may be removed from the cell (i.e. the bond between the recognition pair is broken and the recognition site remains on the surface of the cell), provided that the cell surface is stressed sufficiently to tear or rip a hole in its surface. More preferably the bound recognition pair complex is removed from the cell, optionally together with part of the cell membrane, comprising either the phospholipid bi-layer, other membrane proteins or both.
It is preferred that the recognition agent is attached to the surface of a solid prior to contacting it with the cell. Suitable solids are well known in the art and include particles e.g. beads, and non-particulate materials such as a solid surface of any type such as the surface of sheets, gels, filters, membranes, fibres, capillaries, tubes, plates, dishes and wells. The support may conveniently be made of glass, silica, latex, a polymeric material or a magnetic or magnetisable material. A single recognition agent may be attached to one surface, typically when the solid surface is a bead. However, a plurality of recognition agents may also be attached to the surface of the same solid, such as the surface of a bead, a well, or a test tube.
Particulate solids, especially beads are preferred for use in the methods of the invention. Monodisperse particles, that is those which are substantially uniform in size (e.g. having a diameter standard deviation of less than 5%) have the advantage that they provide very uniform reproducibility of reaction.
When the solid is particulate, for example a bead, a force between the recognition agent and the cell is preferably introduced by perturbing a liquid medium containing the particle. This embodiment will be further described with reference to beads wherein the beads need to be of a size and density such that the force set up between the recognition agent and the cell during perturbation of the liquid medium is sufficient to, for example, separate the cell and the bound recognition pair complex. The density of the bead is preferably 1.3-2.8 g/ml, more preferably 1.4-2.4 g/ml, most preferably 1.4-2.1 g/ml. The bead preferably has a diameter less than the diameter of the cell, more preferably a diameter of less than a third the diameter of the cell and most preferably a diameter of 3-4.5 xcexcm. The type of bead is not particularly limited, provided that it does not adversely affect transfection. The bead is preferably a magnetic or magnetisable bead, or a silica bead.
When the bead is a magnetic or magnetisable bead, any type of magnetic separation can be used. Preferably the beads are first added to the liquid medium containing the cells. The beads are then simply allowed to settle onto a monolayer of cells, and then, after an appropriate period of time (e.g. up to 30 mins) allowing for attachment of the beads to the cells, a magnet is brought into proximity with the liquid medium (e.g. by placing the magnet on top of the tissue culture bottlexe2x80x94the size of the bottle is not particularly limited, provided that the magnet can be brought close enough to the beads to apply sufficient force to the beads) to remove the beads from the cells. In this embodiment, there is no requirement to perturb the liquid medium, since the magnet exerts a force on the magnetic or magnetisable beads pulling them away from the cells. Typically, cell containers can be placed under a magnet for 3-20 minutes, allowing the beads to be pulled away from the cells, thereby creating holes and allowing the substance which it is intended to introduce into the cells to diffuse into the cells.
There is no particular limitation on the ratio of beads to cells used in the present method, but ratios of less than 50:1 are preferred. When the cells are suspension cells, the preferred ratio of beads/cells is any ratio up to 20:1. When the cells are adherent cells, the preferred ratio of beads/cells is any ratio up to 10:1.
In the case where the liquid medium is perturbed, perturbation preferably comprises centrifugation and/or mixing, e.g. mixing by stirring or end-over-end mixing. Perturbation may also include agitation of the liquid in any other manner, provided that sufficient force is set up between the recognition pair complex and the cell to separate the bound recognition pair complex from the cell. Without being bound by theory, it is believed that the perturbation may set up shear forces which separate the bound recognition pair complex from the cell, or forces acting along the axis of bonding between the cell and the recognition agent which separate the bound recognition pair complex from the cell.
When the solid is other than a particulate solid such as a bead, it is preferred that, once the cells are attached to the recognition agents, a liquid medium is allowed to flow across the surface of the solid. Without being bound by theory, it is thought that in this embodiment shear forces are set up which separate the cell from the bound recognition pair complex. In this embodiment the cells are preferably captured on the surface of a test tube or a well and the surface is then washed with a liquid medium. Preferably, the cells are removed from the solid surface by washing them with a fast stream of saline solution containing the substance to be introduced into the cell.
In an alternative embodiment, the recognition agent which binds to the recognition site may not be bound to a solid but may bind to a further agent which is itself bound to a solid. For example, beads may be provided coated with a secondary antibody together with a selection of primary antibodies (recognition agents) targeted to different recognition sites, possibly to different types of cells, all of which primary antibodies bind to the secondary antibody on the beads. Alternatively the recognition agent may be a fusion molecule allowing affinity binding to the further intermediate agent, to allow e.g. streptavidin:biotin binding. In general binding of a recognition agent to a solid support via the intermediacy of one or more further molecules may be achieved using affinity binding or e.g. covalent, hydrophobic or ionic binding. It will be appreciated however that for performance of the invention the association between the recognition agent and the intermediate binding molecule is necessarily stronger than between the recognition agent and the recognition site.
In all of the embodiments employing a liquid medium, it is preferred that the substance to be introduced into the cell is contained within the liquid medium. In this preferred embodiment the substance is introduced into the cell in a step which is simultaneous with the step of separating the recognition agent from the cell. However, it is also possible that the substance can be contacted with the cells once they have been removed from the recognition agent and the transient holes have been created in the cell surface, provided that the substance is introduced before the transient holes in the cell surface re-seal.
Achievement of appropriate separation of the recognition agent from the cell (e.g. by peturbation) may be conveniently assessed by monitoring the extent of transfection, i.e. the number of cells into which the substance to be transfected (e.g. a test molecule) is introduced. Preferably at least 65% of cells, e.g. 75%, more preferably 80% are transfected.
The liquid medium employed is not particularly limited and is preferably an aqueous medium. The medium may be a buffer or a cell culture medium. The concentration of the substance in the medium is not particularly limited and may be selected according to the quantity of substance which is required to be introduced into the cell. As discussed previously, transfection may be optimised by selection of a particular osmolarity for the liquid medium, taking into consideration the osmolarity of the cells to be transfected and whether the cells are adherent or in suspension.
The substance to be introduced can be any substance and will preferably not be endogenous to the cell into which it is to be introduced. Preferably the substance is a substance not normally able to cross the cell membrane. It is preferred that the substance to be introduced into the cell is a hydrophilic substance, however the substance may also be hydrophobic. Any biological molecule or any macromolecule e.g. a complex of molecules can be introduced into the cell. The substance generally has a molecular weight of 100 daltons or more. In a more preferred embodiment, the substance is a nucleic acid molecular such as DNA, RNA, PNA (e.g. cDNA, genomic DNA, a plasmid, a chromosome, an oligonucleotide, a nucleotide sequence, or a ribozyme) or a chimeric molecule or a fragment thereof, or an expression vector. Additionally, the substance may be any bio-active molecule such as a protein, a polypeptide, a peptide, an amino acid, a hormone, a polysaccharide, a dye, or a pharmaceutical agent such as drug. Conveniently the substance to be introduced is present in the liquid medium at a concentration of 0.2 to 10xc3x9710xe2x88x928M, e.g. 0.75-1.25xc3x9710xe2x88x928M.
The cells to which the method of the present invention can be applied are not particularly limited and induce prokaryotic and eukaryotic cells, preferably mammalin e.g. human, cells. It is preferred that the cell against which the method is employed is an animal cell. However, the method can also be employed to treat cells with cell walls, such as plant cells, fungal cells and bacteria. In this latter embodiment, it is preferred that the method is carried out on a protoplast derived from the cell which has had its cell wall partially or completely removed.
Using the method of the present invention, a population of cells can be transfected. These cells may, for instance, be in the form of a cell suspension or may be adherent cells on a solid surface. Suitable solid surfaces include all those discussed previously in connection with the solid to which the recognition agent is attached. Here, however, the solid surface will preferably be a slide, well, dish, flask, plate etc., made conveniently from glass or plastic. The method may also be employed to treat a cell population containing a plurality of cell types. The recognition agent may be specific to a recognition site present on the surface of one or more target cell types in the population, such that the substance is selectively introduced into target cell types within the population.
Thus, in a preferred embodiment of the present invention is provided a method for introducing a non-endogenous nucleic acid molecule into a cell, which method comprises contacting the cell with an antibody attached to a bead, said antibody being able to bind to an antigen on the surface of the cell. When the bead is magnetic or magnetisable, the antibody-coated beads are removed from the cell by introduction of a magnet which attracts the beads away from the cells causing sufficient force to form holes in the cells and thus influx of nucleic acid molecules. When the beads are not magnetic or magnetisable or the magnetic or magnetisable properties of the beads are not to be used in the separation, the liquid medium which contains the cells, nucleic acid material to be introduced and the antibody-coated beads is perturbed by centrifugation or end-over-end mixing to cause separation of the antibody from the cell, hole formation and introduction of the nucleic acid molecule.
The present invention also provides a kit for introducing a substance into a cell, which kit comprises;
(i) a solid surface for attaching to a recognition agent;
(ii) a liquid medium having an osmolarity of from 30-1200 mOsm;
(iii) optionally a recognition agent capable of binding to a recognition site on the surface of a cell to form a recognition pair; and
(iv) optionally, a means for attaching the recognition agent to the solid surface.
The solid surface is not particularly limited, provided that it is suitable for recognition agents to be attached to, and convenient examples have been discussed herein.
In one embodiment in which the kit is suitable for transfecting adherent cells, the osmolarity of the liquid medium provided with the kit is from 20-150, e.g. 40-100 mOsm, more preferably from 40-50 mOsm. In an alternative embodiment in which the kit is suitable for transfecting suspension cells, the osmolarity of the liquid medium provided with the kit is from 800-1000 mOsm, more preferably from 950-1000 mOsm.
The recognition agent can be attached to the solid surface by any means, either direct or indirect, including adsorption onto the surface, covalent linking to the surface, attachment through a nucleic acid linker, or attachment through a linker that is capable of being cleaved chemically or enzymatically. Preferably, the means for attaching the recognition agent to the solid surface is a biotin/avidin coupling, a biotin/streptavidin coupling, or a biotin/modified avidin coupling. The solid surface is preferably coated with avidin and then reacted with a biotinylated antibody. The solid surface is preferably one or more beads.
When the recognition agent is an antibody, the kit may contain a secondary antibody specific to a range of primary antibodies. The kit may thus further contain one or more primary antibodies specific to one or more antigen on the surface of a cell. When a secondary antibody is employed, the secondary antibody is preferably biotinylated to attach it to avidin coated beads. In one variation of the present invention, primary antibodies may first be attached to the cells and then removed by mixing them with beads coated with the secondary antibody. In a further variation, both primary and secondary biotinylated antibodies can be bound to the cells and then mixed with avidin coated beads.
Furthermore, the solid surface may be coated with more than one type of recognition agent to allow the simultaneous transfection of more than one type of cell.
A further example of a kit provided by the invention for introducing a substance into a cell comprises:
(i) a recognition agent capable of binding directly or indirectly to a recognition site on the surface of the cell, said recognition agent optionally being attached to a solid surface;
(ii) optionally a liquid washing medium; and
(iii)optionally a liquid immunoporation medium.
The washing medium, is preferably aqueous and may be a buffer or cell culture medium, preferably an isotonic saline medium of physiologically acceptable pH. The immunoporation medium is a liquid medium as previously defined, e.g. a salt solution which may contain nutrient factors, it will have an osmolarity selected to optimise the introduction of the substrate into the cell in the manner previously discussed. The immunoporation medium in the kit may contain the substance to be introduced into the cell but more preferably this will be added to the immunoporation medium by the person using the kit.
Indirect binding as referred to in part (i) above refers to the situation, for example when the recognition agent is an antibody, that the recognition agent which is bound to a solid surface binds to a factor, e.g. to another antibody which itself binds to the recognition site, e.g. antigen, on the surface of the cell.
When carrying out the method of the present invention, mixing is preferably employed to perturb a liquid medium containing the cell/recognition agent complex. Mixing preferably takes place for a period of from 2-15 hours, more preferably 2-5 hours, most preferably around 3 hours depending on the exact nature of the cell and result required. Longer mixing times may give the best results with certain cell types, for example Human Daudi B cells, originally derived from a patient with Burkitt""s lymphoma, maximum levels of transfection when the cells were mixed with antibody-coated beads for 12-18 hours. Optimal mixing times will depend on the nature of the recognition site and its interaction with the cell membrane and other cellular components, particularly the cytoskeleton.
When an antibody is employed as recognition agent, the antibody used is preferably specific to a protein antigen on the surface of the cells. Alternatively, the antibodies may be specific to other antigen types on the cell surface, such as polysaccharides.
The present invention also provides use of a recognition agent capable of binding to a recognition site on the surface of a cell, in the introduction of a substance into the cell.
The method, kits and use of the present invention can be used in non-medical applications, such as in life-sciences applications, as well as in medical applications.
Life sciences applications in which the present invention can be particularly useful include the introduction of specific genes into viable cells for expression and for the analysis of the effect of gene products on the metabolism of cells. Appropriately transfected cells may also be used to express useful products which may be harvested, e.g. human insulin. Such applications also include the introduction of biologically active proteins into viable cells to study their effects on the cells with regard to the metabolism and morphology of the cells. These applications also extend to the introduction of pharmacologically important compounds into cells, where the cell membrane is normally impervious to such compounds. Cells transfected according to the method described herein form a further aspect of the invention.
Medical applications in which the present invention can be particularly useful include all applications which are not life sciences applications as defined above, and specifically include gene therapy and the targeted introduction of new genes into specific immunologically defined cell populations with deleted or defective genes. Such applications also include the introduction of genes controlling endocrine functions, such as hormone synthesis and secretion, into epidermal cell populations. The targeted permeabilisation of tumour cells for enhanced chemotherapy procedure is also included in these applications. In particular, the method kit and use of the present invention may be employed in gene therapy to great advantage. For example, they may be used in the treatment of leukaemic cells, such cells being treated in suspension in preference to non-leukaemic cells present in the same suspension. Additionally, the invention may be used to introduce antisense RNA into specific cell types. The method may be used ex vivo, on cells in body fluids, tissue or organs, which may be reintroduced into the body, or in vivo
In comparison to known methods, the present method is very efficient. The efficiency i.e. the proportion of cells into which the target substance has been introduced, depends, inter alia, on the length of time during which mixing is carried out and the vigorousness of the mixing. Efficiency of at least 70%, preferably at least 80%, more preferably at least 90% can be achieved and in some circumstances, an efficiency of 100% can be approached. In embodiments where there is no mixing as such, e.g. where the cells are adherent and separation occurs as a result of magnets pulling magnetic or magnetisable beads away from the cells, efficiencies of at least 85%, preferably 90% or more can readily be achieved.